Many plant species, including Nicotiana alata and Lycopersicon peruvianum, are self-incompatible, that is they cannot be fertilized by pollen from themselves or by that of a plant of the same S- (or self-incompatibility) genotype. The molecular basis of self-incompatibility is believed to arise from the presence of S-protein in the mature styles of plants; in particular, as exemplified by N alata and L. peruvianum, S-protein has now been shown to be present in extracts of plant styles at the developmental stages of buds at first show of petal color, and at the subsequent stages of maturation of open but immature flowers, and flowers having mature glistening styles. 0n the other hand, S-protein is not present in the earlier developmental stages of green bud and elongated bud.
For general reviews of self-incompatibility, see de Nettancourt (1977) Incompatibility in Angiosperms, Springer-Verlag, Berlin; Heslop-Harrison (1978) Proc. Roy. Soc. London B, 202:73; Lewis (1979) N. Z. J. Bot. 17:637; Pandey (1979) N.Z. J. Bot. 17:645 and Mulcahy (1983) Science 220:1247. Self-incompatibility is defined as the inability of female hermaphrodite seed plants to produce zygotes after self-pollination. Two types of self-incompatibility, gametophytic and sporophytic, are recognized. Gametophytic incompatibility is most common and in many cases is controlled by a single nuclear gene locus (S-locus) with multiple alleles. Pollen expresses its haploid S-genotype and matings are incompatible if the S-allele expressed is the same as either of the S-alleles expressed in the diploid tissue of the pistil. During both incompatible and compatible matings, pollen tubes germinate and grow through the stigma into the transmitting tissue of the style. Tube growth from incompatible pollen grains is arrested in the upper third of the style.
In sporophytic incompatibility, pollen behavior is determined by the genotype of the pollen-producing plant. If either of the two S-alleles in the pollen parent is also present in the style, pollen tube growth is inhibited. Unlike the gametophytic systems, inhibition usually occurs at the stigma surface and not in the style. In sporophytic incompatibility, S-protein may be concentrated at or near the stigma surface. The gametophytic polyallelic system is considered to be the ancestral form of self incompatibility in flowering plants with the sporophytic system being derived from it (de Nettancourt 1977, supra). The products of the S-gene in the two systems are considered to be structurally related.
There are five species of gametophytically self-incompatible plants and two species of sporophytically incompatible plants in which style or stigma proteins apparently related to S-genotype have been detected by either electrophoretic or immunological methods. In alata, an association between specific protein bands and three S-allele groups was demonstrated by isoelectric focussing of stylar extracts (Bredemeijer and Blaas (1981) Theor. Appl. Genet. 59:185). Two major antigenic components have been identified in mature styles of a Prunus avium cultivar of S.sub.3 S.sub.4 genotype, one of which (S-antigen) was specific to the particular S-allele group (Raff, et al. (1981) Planta 153:125; and Mau, et al. (1982) Planta 156:505). The S-antigen, a glycoprotein, was a potent inhibitor of the in vitro growth of pollen tubes from a S.sub.3 S.sub.4 cultivar (Williams et al. (1982) Planta 156:577). The glycoprotein was resolved into two components, purportedly representing the S.sub.3 and S.sub.4 products of the S.sub.3 S.sub.4 genotype. Stylar protein components which have been associated with the S-allele group or the self-incompatibility genotype are reported in Petunia hybrida (Linskens (1960) Z. Bot. 48:126), Lilium longiflorum and Trifolium pratens (Heslop-Harrison (1982) Ann. Bot. 49:729).
A glycoprotein corresponding to genotype S.sub.7 of Brassica campestris has been isolated from extracts of stigmas by gel-filtration followed by affinity chromatography and isoelectric focussing (Nishio and Hinata (1979) Jap. J. Genet. 54:307). Similar techniques were used to isolate S-specific glycoproteins from stigma extracts of Brassica oleracea plants homozygous for S-alleles S.sub.39, S.sub.22 and S.sub.7 (Nishio and Hinata (1982) Genetics 100:641). Antisera raised to each isolated S-specific Brassica oleracea glycoprotein not only precipitated its homologous glycoprotein but also reacted with the other two S-specific glycoproteins of B. oleracea and the S.sub.7 -specific glycoprotein of B. campestris (Hinata et al. (1982) Genetics 100:649). An S-specific glycoprotein was isolated by Ferrari et al. (1981) Plant Physiol. 67:270 from a stigma extract of B. oleracea using sucrose gradient sedimentation and double diffusion tests in gels in which the proteins were identified by Coomassie Blue staining. This preparation was shown to be biologically active since pretreatment of S.sub.2 S.sub.2 pollen with the glycoprotein prevented the pollen from germinating on normally compatible stigmas. Recently a cDNA clone encoding part of an S-locus specific glycoprotein from B. oleracea stigmas has been described (Nasrallah et al. (1985) Nature 318:263-267.
In work that is detailed in Clarke et al., U.S. patent applications Ser. No. 615,079, filed May 24, 1984, and Ser. No. 050,747, filed May 15, 1987, stylar extracts of several self-incompatibility genotypes from both Nicotiana alata and Lycopersicon peruvianum were examined for the presence of S-gene associated protein. Glycoprotein materials were identified in the 30,000 MW region of stylar extracts of genotypes S.sub.1 S.sub.3, S.sub.2 S.sub.3, S.sub.2 S.sub.2 and S.sub.3 S.sub.3 of N. alata and of genotypes S.sub.1 S.sub.2, S.sub.2 S.sub.3, S.sub.1 S.sub.3, S.sub.2 S.sub.2, S.sub.3 S.sub.3 and S.sub.3 S.sub.4 of L. peruvianum. By comparing two-dimensional gel electrophoresis of stylar extracts of the different genotypes, closely related, but distinct glycoproteins were found to segregate with the individual S-alleles. For example, the N. alata S.sub.2 -protein was found only in stylar extracts of the genotypes containing the S.sub.2 -alleles (S.sub.2 S.sub.3 and S.sub.2 S.sub.2). For each genotype, the genotype specific glycoprotein only appeared as the flower matured, and was detected only in stylar extracts of buds at first show of petal color and in later stages of maturation, but not in earlier bud stages. Therefore, the appearance of these glycoproteins is temporally coincident with the appearance of the self-incompatibility phenotype. The S.sub.2 -glycoprotein of N. alata and the S.sub.2 and S.sub.3 -proteins of L. peruvianum were shown to be more highly concentrated in the upper style sections, which is the zone in which pollen tube inhibition occurs. Therefore, the appearance of these glycoproteins is spatially coincident with the self-incompatibility reaction. Further, corroboration of the biological activity of S.sub.2 -protein of N. alata was demonstrated by its inhibition of pollen tube growth in an in vitro assay (Williams, et al., 1982, supra).
A significant aspect of the work disclosed in U.S. application Ser. Nos. 615,079 and 050,747 was the discovery that rabbit antisera and monoclonal antibodies raised to individual S-proteins or stylar extracts showed immunological cross-reaction between S-proteins of different genotype within the same species, between S-proteins of different species and also between species having gametophytic incompatibility and sporophytic incompatibility. It was concluded therein that there is structural homology among S-proteins, and that despite apparent differences in molecular weight and pI, these proteins are a recognizable structural class in addition to their functional similarities.
These applications also reported the results of N-ter,oma; sequencing of several mature N. a;ata (S.sub.2, S.sub.6, S.sub.Z and S.sub.f11) proteins and L. peruvianum (S.sub.1 and S.sub.3) proteins. Significant amino acid sequence homologies among these gametophytic S-proteins were found. In the region sequenced (amino acids 1-15), the N. alata S.sub.2 protein is 80% homologous to the N. alata S.sub.6 protein, 67% homologous to the L. peruvianum S.sub.1 protein, 53% homologous to the L. peruvianum S.sub.3 protein.
U.S. application Ser. Nos. 615,079 and 050,747 also disclosed a method of purification for S-proteins which included fractionation of stylar extracts by ion exchange chromatography followed by a second fractionation by affinity chromatography. The method of purification was exemplified with the isolation of the 32K S.sub.2 -glycoprotein from Nicotiana alata styles.
Recent reports of the isolation and amino acid sequence of the S.sub.8, S.sub.9 and S.sub.12 proteins of Brassica campestris show that there is extensive homology among these gametophytic S-proteins (Takayama et al. (1986) Agric. Biol. Chem. 50:136501367; Takayama et al. (1986)
ibid. p. 1673-1676; Takayama et al. (1987) Nature 326:102-105). The predicted amino acid sequence of the S.sub.6 protein of B. oleracea (Takayama et al., 1987, supra) based on the DNA sequence of an S.sub.6 gene cDNA clone (Nasrallah et al., 1985, supra) is found to be about 75% homologous to the B. campestris S-proteins. Comparison of the N. alata and L. peruvianum S-protein sequences (U.S. patent applications Ser. No. 615,079 and 050,747; Anderson et al. (1986) Nature 321:38-44) with those of the Brassica S-proteins indicate that there is no significant homology between the gametophytic and sporophytic S-proteins.
The S-proteins that have been identified are glycoproteins, which are proteins that have been modified by covalent bonding of one or more carbohydrate groups. Little is known of the composition and structure of the carbohydrate portion of S-proteins. It is, as yet, unclear what contribution, if any, the carbohydrate portion of the S-protein makes to biological activity in the incompatibility reaction. Petunia hybrida stylar mRNA is translated in Xenopus laevis (frog) egg cells to produce active proteins which induce the incompatibility reaction. The relative glycosylation of S-proteins produced in frog egg cells to that of the S-proteins produced in the plant is unknown; however, the post-translational processing in the foreign system is adequate to produce biologically active proteins (Donk, van der J. A. W. M., (1975) Nature 256:674-675).
Most proteins, such as the S-proteins, that are excreted from or transported within cells have signal or transit sequences that function in the translocation of the protein, for example see: Perlman, D. and Halverson, H. W., (1983) J. Mol. Biol. 167:391-409; Edens, L. et al. (1984) Cell 37:629-633.; and Messing, J. et al. in Genetic Engineering of Plants, ed. Kosuge, T. et al. (1983) Plenum Press, New York, pp. 211-227. Signal or transit DNA sequences are generally adjacent to the 5' end of the DNA encoding the mature protein, are co-transcribed with the mature protein DNA sequence into mRNA and are co-translated to give immature proteins with the signal or transit peptide attached. During the translocation process the signal or transit peptide is cleaved to produce the mature protein.
The expression of S-genes in self-incompatible plants shows very complex regulation, with S-gene products appearing in only certain tissues at certain times. The mechanism of this regulation is not yet known in detail, but involves the presence of specific regulatory DNA sequences in close proximity to the genomic DNA that encodes the S-protein. Adjacent to the structural gene and signal or transit sequences, are promoter sequences that control the initiation of transcription and exert control over protein expression levels.